title: Efavirenz stimulates HIV-1 reverse transcriptase RNase H activity by a mechanism involving increased substrate binding and secondary cleavage activity.
abstract: Efavirenz is a non-nucleoside reverse transcriptase inhibitor used for treating HIV/AIDS. We found that polymerization activity of a reverse transcriptase (RT) with the E478Q mutation that inactivates the RNase H catalytic site is much more sensitive to efavirenz than wild-type RT, indicating that a functional RNase H attenuates the effectiveness of efavirenz. Moreover, efavirenz actually stimulated wild-type RNase H binding and catalytic functions, indicating another link between efavirenz action and RNase H function. During reverse transcription in vivo, the RT that is extending the DNA primer also periodically cleaves the genomic RNA. The RNase H makes primary template cuts ~18 nucleotides from the growing DNA 3'-end, and when the RT pauses synthesis, it shifts to make secondary cuts ~9 nucleotides from the DNA 3'-end. After synthesis, RTs return to bind the remaining template RNA segments at their 5'-ends and make primary and secondary cuts, 18 and 9 nucleotides in, respectively. We found that efavirenz stimulates both 3'- and 5'-directed RNase H activity. Use of specific substrates revealed a particular acceleration of secondary cuts. Efavirenz specifically promoted binding of the RT to RNase H substrates, suggesting that it stabilizes the shifting of RTs to make the secondary cuts. We further showed that efavirenz similarly stimulates the RNase H of an RT from a patient-derived virus that is highly resistant and grows more rapidly in the presence of low concentrations of efavirenz. We suggest that for efavirenz-resistant RTs, stimulated RNase H activity contributes to increased viral fitness.
authors.affiliation: Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry , Rochester, New York 14642, United States.
authors.forename: John M
authors.initials: JM
authors.lastname: Muchiri
authors.forename: Dongge
authors.initials: D
authors.lastname: Li
authors.forename: Carrie
authors.initials: C
authors.lastname: Dykes
authors.forename: Robert A
authors.initials: RA
authors.lastname: Bambara
mesh_terms: D000480:Alkynes; D001483:Base Sequence; D048588:Benzoxazines / Q000494:pharmacology*; D003521:Cyclopropanes; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D054303:HIV Reverse Transcriptase / Q000378:metabolism*; D059748:Proteolysis; D018894:Reverse Transcriptase Inhibitors / Q000494:pharmacology*; D016914:Ribonuclease H / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D000480:Alkynes; D048588:Benzoxazines; D003521:Cyclopropanes; D017931:DNA Primers; D018894:Reverse Transcriptase Inhibitors; C514824:reverse transcriptase, Human immunodeficiency virus 1; D054303:HIV Reverse Transcriptase; D016914:Ribonuclease H; C098320:efavirenz